Leishmania is a group of parasitic protozoa that infect human macrophages and thrive inside the hostile environment of the phagolysosomes of these cells. The long-term objective of the proposed research is to identify the molecular events that must occur at the early stage of leishmanial interactions with the macrophages leading to the establishment of successful parasitism. Leishmania has long been known to 'renovate' the molecular environment of macrophages in order to establish infection inside their phagolysosomes. Leishmania specifically manipulates the expression of host macrophage genes in the early stages of their infection. Gene transcripts that are down regulated in macrophages upon exposure to Leishmania include 7SL RNA, the RNA component of the signal recognition particle (SRP). Since the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down regulation of 7SL RNA may be significant in the establishment of infection by Leishmania. More importantly, over expression of 7SL RNA in J774G8 or U937 cells confers resistance to Leishmania infection. These results demonstrate the biological significance of down-regulating 7SL RNA synthesis in the establishment of infection by Leishmania. Based on these findings, the hypothesis is that Leishmania-induced down regulation of the level of 7SL RNA in the macrophages favors in part the development of leishmaniasis in the mouse model. Exogenous compensation of 7SL RNA in their macrophages will thus make these mice resistant to Leishmania infection.
Specific aims to test the hypothesis are the following: (1) Development of BALB/c mice derivatives with over expression of 7SL RNA in their macrophages by genetic manipulation of the bone marrow stem cells and transplantation. Over expression of 7SL RNA in the bone marrow derived macrophages will be done using already developed lentiviral constructs that will allow the expression to occur inside the cells of macrophage lineage. Genetically manipulated bone marrow stem cells will be transplanted into irradiated BALB/c mice, which are susceptible hosts for Leishmania. (2) Evaluation of the ability of Leishmania promastigotes to produce footpad lesions in mice over expressing 7SL RNA in their macrophages. (3) Evaluation of the effects of Leishmania exposure of macrophages isolated from the peritoneum of the transplanted mice on the levels of phagolysosomal proteins, surface membrane receptors and the levels of proteins secreted from these cells. We will measure the levels of cathepsins in lysosomes, and scavenger receptor, and CSF-1R on the cell surfaces of peritoneal macrophages isolated from transplanted mice after exposure of the macrophages for various time periods (0-12 h). These evaluations will be done by immunofluorescence microscopy and Western blotting analysis. Levels of cytokines and chemokines secreted from the macrophages with or without Leishmania exposure will be evaluated by cytokine antibody microarray analysis.
The proposed study will reveal a unique renovating mechanism employed by the parasitic protozoan Leishmania to establish infection in the macrophages. Development of the mouse model with over expression of 7SL RNA in its macrophages may be used to test whether macrophage vesicular protein transport is also critical for the parasitism of macrophages by other pathogens. Understanding host-parasite interaction mechanisms interplayed between macrophages and Leishmania in molecular details will help us in developing combat measures against this often deadly human pathogen. ? ? ?
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